Diagnostic method and means therefor

ABSTRACT

Indirect hemagglutination is utilized in a serological method to detect the presence of a bacterial infection by means of antibacteriophage antibodies in the serum of a patient. The antibodies are caused to react with known bacteriophages which are fixed on aldehyde-treated erythrocytes and the resulting hemagglutination serves as an indicium of the types of antibodies that are present. Bacteriophages are adsorbed on aliphatic aldehyde-treated erythrocytes at a temperature of at least about 45* C. and fixed by a subsequent aliphatic aldehyde treatment.

United States Patent [191 Kamme 51 Oct. 22, 1974 [75] Inventor: Carl Gustaf Kamme, Lund, Sweden [73] Assignee: Aktiebolaget Leo (A/B Leo),

Halsingborg, Sweden 22 Filed: May 6,1971

21 Appl. No; 141,026

[52] 11.5. C1. 424/12 [51] Int. Cl. G0ln 31/00 [58] Field of Search 424/12 [56] References Cited UNITED STATES PATENTS 3,548,051 12/1970 Dingwall 424/12 3,590,116 6/1971 Matisheck et a1. 424/12 FOREIGN PATENTS OR APPLICATIONS 1,272,020 7/1968 Germany 424/12 OTHER PUBLICATIONS PSEBM, 124:2, 267, 344-347.

Appl. Microbiol, Vol. 17, No. 3, (1967), pp. 422425.

Primary Examiner-Albert T. Meyers Assistant ExaminerFrederick E. Waddell Attorney. Agent, or FirmGordon W. Hueschen I 5 7 ABSTRACT Indirect hemagglutination is utilized in a serological method to detect the presence of a bacterial infection by means of antibacteriophage antibodies in the serum of a patient. The antibodies are caused to react with known bacteriophages which are fixed on aldehydetreated erythrocytes and the resulting hemagglutination serves as an indicium of the types of antibodies that are present. Bacteriophages are adsorbed on aliphatic aldehyde-treated erythrocytes at a temperature of at least about 45 C. and fixed by a subsequent aliphatic aldehyde treatment.

9 Claims, No Drawings 1 DIAGNOSTIC METHOD AND MEANS THEREFOR BACKGROUND OF THE INVENTION In the case of many deep-seated bacterial infections, and in particular staphylococcal infections, it is difficult or even impossible to obtain a diagnosis using a bacterial culture.

As a complement to bacterial cultures the serological method presently employed is directed to the detection of serum antibodies that arise in response to the presence of a hemolyzing enzyme which is produced by the infecting bacteria. In order to detect and diagnose a staphylococcal infection the so-called antistaphylolysin test (ASta) is used. However, this test gives a rather strong positive reaction in about percent of healthy individuals and a definite positive reaction in only about 30 percent of individuals having such infections. Thus the need for a better serological reaction, in staphylococcal infections, is readily apparent.

Indirect hemagglutination as a sensitive means for tracing vital antibodies is known, however it has not been applied as a diagnostic means for detecting the presence of antibodies to phages. Such means involves the agglutination of erythrocytes that takes place when viral antibodies react with antigens that have been absorbed on the surfaces of erythrocytes. The adsorption of pathogenic virus to erythrocytes pre-treated with tannic acid in buffered saline solutions is disclosed by Friedman et al., Proc. Soc. Exp. Biol. & Med. 74, 712 (1957) and also by Felton et al., J. Immun. 86, 42 1961). However, the foregoing procedure is not applicable to detecting bacteriophage antibodies because the phages are not absorbed to erythrocytes in buffered saline at any pH. Moreover, the antigen-erythrocyte complex is not very stable and cannot be preserved for extended periods of time without a deterioration in properties. For a practical application and ready availability of such a diagnostic method this is of paramount importance, of course.

Accordingly, it is an object of the present invention to provide a diagnostic method and means for detecting the presence of a bacterial infection.

It is a further object of this invention to provide a method for producing a diagnostic means comprising a stable phage-erythrocyte complex having unimpaired antigenic sites of the bacteriophages.

Still other objects will readily present themselves to one skilled in the art upon reference to the ensuing specification and claims.

SUMMARY OF THE INVENTION The present invention contemplates a diagnostic method for bacterial infection utilizing indirect hemagglutination to test for the presence of antibacteriophage antibodies. The diagnostic method comprises admixing of an aliquot of serum from a living animal body with suspected bacterial infection with erythrocytes carrying known bacteriophages fixed thereon. If there are present serum antibodies toward the phages carried by the erythrocytes, hemagglutination will take place thereby indicating that a bacterial infection is present in the living animal body, the phages indigenous to the bacteria being of a type similar to those carried by the erythrocytes.

The phage-bearing erythrocytes are produced by first contacting the erythrocytes with an aliphatic aldehyde,

preferably formaldehyde, and thereafter contacting therewith, in a suitable medium, the bacteriophages at a temperature above about 45 C., preferably in the range of about 50 C. to about 60 C., and in the presence of a sugar such as glucose, or a protein such as peptone, serum globulin or albumin. The bacteriophages adhering to the erythrocytes are fixed thereon by a subsequent aldehyde treatment. In a preferred embodiment of the present invention the aldehyde treated erythrocytes are further treated with tannic acid, i.e., tanned, prior to the addition of bacteriophages.

DESCRIPTION OF THE PREFERRED EMBODIMENTS Preparation of Phage-Bearing Erythrocytes Erythrocytes for use as carriers of bacteriophages in accordance with the present invention can be derived from any suitable source. Sheep erythrocytes, appropriately washed, are preferred; however, rabbit eryth rocytes, human erythrocytes, and the like can also be employed.

Suitable phages are obtained by growing a culture of the propagating strain of bacteria and allowing the culture to reach a predetermined point in the logarithmic growth curve which is different for each type of phage. At this predetermined point a known amount of phage inoculum'is added which immediately begins to attack the bacterial cells by penetrating the cell membrane, multiplying within bacterial cells, and within a few hours causing disintegration or lysis of these cells. Phages liberated from the cells in such a manner remain suspended inthe growth medium. The growth medium is then centrifuged and filtered so as to remove the heavier cell fragments leaving a clear broth containing the phages.

The erythrocytes are first treated with an aqueous solution of an aliphatic aldehyde, such as formaldehyde. Preferably the aldehyde concentration is in the range of about 0.5 to about 5 percent by volume, and more preferably about 1 percent by volume. Solution temperature is not critical, however the temperature should not be so high as to hemolyze the erythrocyte. Typical aliphatic aldehydes, other than formaldehyde, that can be employed are those having up to about five carbon atoms such as glutaraldehyde, pyrovic aldehyde, and the like.

In order to promote the adherence of a bacteriophage on the erythrocyte surface, particularly when the erythrocyte has been treated with formaldehyde, it is desirable to contact the aldehyde-treated erythrocyte with an adherence-promoting agent, such as an aqueous tannic acid solution, for a time period of about 15 minutes to about 60 minutes, preferably about 30 minutes. The tannic acid concentration can range from about 0.1 to about 0.0001 percent by weight, and preferably from about 0.01 to about 0.001 percent. Diazotized benzidine can be used in lieu of tannic acid, if desired, in similar concentrations.

If pyruvic aldehyde is used to treat the erythrocytes, the tannic acid treatment can be normally omitted.

Adherence of a bacteriophage to an erythrocyte prepared in the aforedescribed manner is achieved at an elevated temperature of at least about 45 C. and preferably at a temperature of about 50 C. to about 60 C. in the presence of a sugar such as glucose or a protein such as peptone, preferably in an amount of about 0.05 to about 5 percent by weight, based on the amount of v pended in saline at an erythrocytes present. Usually an aqueous suspension of the erythrocytes is admixed with an aqueous suspension containing the desired bacteriophages and incubated for a time period of about 3 to about 60 and preferably about 20 minutes.

The bacteriophages are then fixed on the erythrocytes by a subsequent treatment with an aqueous aliphatic aldehyde solution preferably at a temperature of about 35C. to about 60 C. and more preferably of about 40 C. to about 50 C. Temperatures greatly exceeding 60 C. are undesirable because changes in the nature of the phage proteins may be caused. The aldehyde concentration in the solution can vary, but preferably is in the range of about 0.5 to about 5 percent by volume and more preferably about 1 percent by volume. Fixation time at the foregoing temperatures can range from about minutes to about 10 hours, depending on the particular bacteriophage that is adsorbed. Usually a fixation time of about 1 hour is sufficient.

The thus prepared phage-bearing erythrocytes can be stored for extended periods of time either as aqueous suspensions, or as lyophilized dry materials which are then reconstituted prior to use.

The preparation of erythrocytes suitable as diagnostic means and having bacteriophages fixed thereon is illustrated by the following examples.

EXAMPLE 1: Formalinization Sheep blood, collected in Alsevers solution, Bukantz et al., J. Lab. Clin. Med. 3, 394 (1946), and stored at 4 C. is centrifuged and erythrocytes recovered therefrom. The erythrocytes are then washed three times by centrifugation in 0.9 percent (w/v) saline and susetythrocyte concentration of about 8 percent.

Equal parts of the 8 percent erythrocyte suspension in saline and a 3 percent (w/v) formaldehyde solution are admixed and the resulting mixture incubated at 37 C. for about 18 to 24 hours. After incubation the erythrocytes are recovered by centrifugation, washed three times, and resuspended in saline to an erythrocyte concentration of 10 percent. Sodium ethylmercurithiosalicylate (Merthiolate) is then added to the erythrocyte suspension to a final concentration of l/ 10,000 (w/v) to prevent growth of microorganisms, and the erythrocytes stored at 4 C.

EXAMPLE 2: Tannic Acid Treatment Erythrocytes formalinized as in Example 1 are washed three times in 0.15 mol phosphate buffered saline (pH 6.4) and resuspended to a concentration of 5 percent. Equal parts of the 5-percent suspension of erythrocytes and a tannic acid solution are then combined and incubated at 56 C. for 30 minutes. The final concentration of tannic acid is usually l/40,000 (w/v). After the incubation period the erythrocytes are again washed three times in the aforementioned buffer. EXAMPLE 3: Fixation of Phages to the Tanned Erythrocytes An aqueous broth containing phages is admixed with erythrocytes treated in the manner set forth in Examples 1 and 2. The resulting admixture is then incubated at about 56 C. for minutes and then cooled to room temperature. Phage-coated erythrocytes are recovered from the admixture by centrifugation and made up into a l to 2 percent suspension in a l-percent .(w/v) aqueous formaldehyde solution. This suspension is then incubated at about 45 C. for 45 minutes, the erythrocytes recovered by centrifugation, washed in buffered saline, and resuspended to a desired concentration for storage. Diagnostic Method The diagnostic method using erythrocytes prepared in the foregoing manner comprises the addition thereof to an aliquot of serum from a living animal body suspected of suffering from a bacterial infection. Hemagglutination of the erythrocytes is indication that phage antibodies are present in the serum. in the case of staphylococcal infections about phages derived from bacteria of the genus Staphylococcus have been grouped serologically with regard to their antigenic structure by means of the indirect hemagglutination test. it was found that all of these phages can be classified into one of four groups, i.e., C1, C2, C3 and C4.

Phages within the same group are antigenically identicytes. The higher the dilution at which hemagglutination occurs, the greater is the antibody count in the serum.

EXAMPLE 4: Comparison of the Antistaphylolysin (ASta) andAntistaphylophage (ASPA) Reactions ASta Reaction Serum aliquots derived from a patient are serially diluted with saline and placed in separate tubes. A predetermined amount of staphylolysin with known strength expressed in international units is added to each tube, the tubes incubated at 37 C. for about 30 minutes, and then a 2 weight percent suspension of washed rabbit erythrocytes is added thereto. The tubes are then further incubated for about 1 hour at 37 C. and for about 3 to 16 hours at 4C. Thereafter the number of antitoxin (antistaphylolysin) units present are determined by ascertaining the degree of erythrocyte hemolysis. Toxin hemolyzes the erythrocytes that are present, but the antitoxin present in the patients serum neutralizes the action of the toxin. Thus, the higher the amount of antitoxin in the serum, the more the serum can be diluted while still retaining toxin neutralization ability. The amount in the serum is expressed in units.

ASPA Reaction:

Aqueous suspensions of sheep erythrocytes with staphylococcal phages fixed thereon, each suspension representing one of the main serological groups according to their capsids, are provided. Such phagecoated erythrocytes can be stored for at least about 8 months at 4 C. without deterioration.

Serum aliquots from a patient are admixed with uncoated erythrocytes and the resulting admixture is kept at about room temperature for about 1 hour in order to remove from the serum heterophilic antibodies, i.e., such antibodies that are active toward the erythrocytes. Thereafter the erythrocytes are separated from the serum by centrifugation and serial two-fold dilutions of the serum, sans heterophilic antibodies, are admixed with a known amount of the phage-coated erythrocytes in small tubes or cups having spherical bottoms. Upon admixture and incubation hemagglutination takes place in the presence of antiphage antibodies. The hemagglutination reaction takes place at about room temperature in about two hours. The more the serum aliquot can be diluted and still result in hemagglutination, the more antibodies (higher titer) are present in a patients serum. Comparison of the Reactions Serum derived from patients with deep-seated staphylococcal infections, which were verified by cultivation of staphylococcal strains from their lesions, was divided into aliquots and subjected to both the ASta and the ASPA tests as set forth above. The test data are presented in Table 1 below. Values of the ASPA titers are given. Limit Value (ASta):

2.2 units, i.e., 2.2 negative and 2.2 positive Limit Value (ASPA):

titer 1/40, i.e., l/40 negative and l/40 positive From the above data it is readily apparent that the diagnostic method of the present invention (the ASPA test) is much more sensitive. Using the ASta test a negative indication was obtained for six out of 10 infected patients whereas with the ASPA test a positive indication was obtained in all instances.

The foregoing discussion and the examples are to be taken as illustrative of, but not limiting, the present in vention. Still other variations within the spirit and scope of the present invention will readily present themselves to one skilled in the art.

1 claim:

1. A method in which erythrocytes bearing phages derived from bacteria are produced which comprises contacting erythrocytes with an aqueous C to C aliphatic aldehyde solution. contacting the resulting alde- 6 hyde-treated erythrocytes with Staphylococcal bacteriophages at a temperature of about 45 C to about 60 C for a period of time sufficient to adhere said phages to said erythrocytes, and then contacting the phagebearing erythrocytes with an aqueous C to C 5 aliphatic aldehyde solution at a temperature of about 35 C to about 60 C for a period of time sufficient to fix said phages to said erythrocytes.

2. The method of claim 1 in which the aldehydetreated erythrocytes are contacted with the bacteriophages in the presence of a sugar or protein selected from the group consisting of glucose. peptone, serum globulin. and albumin.

3. The method of claim 1 in which the aldehydetreated erythrocytes are contacted with the bacteriophages in the presence of the broth from the nutrient medium in which the phage is produced.

4-. The method of claim 1 wherein the erythrocytes are derived from sheep and the aldehyde is formaldehyde.

5. The method of claim 1 wherein the contacting of the aldehyde-treated erythrocytes with a phage is carried out at a temperature of about 50 to about 60 C.

6. The method of claim 1 wherein the aldehydetreated erythrocytes are further contacted with tannic acid prior to the contact with a phage.

7. Erythrocytes having Staphylococcal phages fixed thereon, prepared by the method of claim 1.

8. A method of diagnosis for a bacterial infection caused by bacteria of the genus Staphylococcus having bacteriophages indigenous thereto, which comprises providing an aliquot of serum from a living animal body, pre-treating said serum with erythrocytes so as to remove heterophilic antibodies from the serum, and admixing said pre-treated serum with erythrocytes having a phage of a type known to be indigenous to bacteria of the genus Staphylococcus fixed thereon prepared by the method of claim 1 in amounts sufficient to cause hemagglutination of the erythrocytes if antibodies active against said phage are present in said serum, the agglutination serving to indicate that a bacterial infection by bacteria having indigenous phages of a similar type-is present in said living animal body.

9. The diagnostic method of claim 8 wherein an aliquot of the serum is serially diluted and the diluted portions thereof are admixed with known amounts of said phage-bearing erythrocytes. 

1. A METHOD IN WHICH ERYTHROCYTES BEARING PHAGES DERIVED FROM BACTERIA ARE PRODUCED WHICH COMPRISES CONTACTING ERYTHROCYTES WITH AN AQUEOUS C1 TO C5 ALIPHATIC ALDEHYDE SOLUTION, CONTACTING THE RESULTING ALDEHYDE-TREATED ERYTHROCYES WITH STAPHYLOCOCCAL BACTERIOPHAGES AT A TEMPERATURE OF ABOUT 45* C TO ABOUT 60*C FOR A PERIOD OF TIME SUFFICIENT TO ADHERE SAID PHAGES TO SAID ERYTHROCYTES, AND THEN CONTACTING THE PHAGEBEARING ERYTHROCYTES WITH AN AQUEOUS C1 TO C5 ALIPHATIC ALDEHYDE SOLUTION AT A TEMPERATURE OF ABOUT 35*C TO ABOUT 60*C FOR A PERIOD OF TIME SUFFICIENT TO FIX SAID PHAGES TO SAID ERYTHROCYTES.
 2. The method of claim 1 in which the aldehydetreated erythrocytes are contacted with the bacteriophages in the presence of a sugar or protein selected from the group consisting of glucose, peptone, serum globulin, and albumin.
 3. The method of claim 1 in which the aldehydetreated erythrocytes are contacted with the bacteriophages in the presence of the broth from the nutrient medium in which the phage is produced.
 4. The method of claim 1 wherein the erythrocytes are derived from sheep and the aldehyde is formaldehyde.
 5. The method of claim 1 wherein the contacting of the aldehyde-treated erythrocytes with a phage is carried out at a temperature of about 50* to about 60* C.
 6. The method of claim 1 wherein the aldehyde-treated erythrocytes are further contacted with tannic acid prior to the contact with a phage.
 7. Erythrocytes having Staphylococcal phages fixed thereon, prepared by the method of claim
 1. 8. A method of diagnosis for a bacterial infection caused by bacteria of the genus Staphylococcus having bacteriophages indigenous thereto, which comprises providing an aliquot of serum from a living animal body, pre-treating said serum with erythrocytes so as to remove heterophilic antibodies from the serum, and admixing said pre-treated serum with erythrocytes having a phage of a type known to be indigenous to bacteria of the genus Staphylococcus fixed thereon prepared by the method of claim 1 in amounts sufficient to cause hemagglutination of the erythrocytes if antibodies active against said phage are present in said serum, the agglutination serving to indicate that a bacterial infection by bacteria having indigenous phages of a similar type is present in said living animal body.
 9. The diagnostic method of claim 8 wherein an aliquot of the serum is serially diluted and the diluted portions thereof are admixed with known amounts of said phage-bearing erythrocytes. 